Selenium yeast promoted the Se accumulation, nutrient quality and antioxidant system of cabbage (Brassica oleracea var. capitata L.)

ABSTRACT

The application of Se yeast as a Se source to cultivate Se-rich cabbage has a significant effect on cabbage growth and quality indices. Results showed that total plant weight, head weight, and head size in cabbage were notably increased by 48.4%, 88.3%, and 25.4% under 16 mg/kg Se yeast treatment, respectively. Compare with the control, a high proportion of 3874% of Se accumulation in cabbage head was also detected in 16 mg/kg Se yeast treatment. Selenocystine (SeCys₂) and Methyl-selenocysteine (MeSeCys) were the main Se speciations in the cabbage head. Application of 8 mg/kg Se yeast improved cabbage quality and antioxidant system indices, including free amino acid, soluble sugar, ascorbic acid, phenolic acid, glucosinolates, and SOD activity, which had 81.6%, 46.5%, 34.9%, 12.3%, 44.8%, 25.2% higher than that of the control, respectively. In summary, considering 8 mg/kg Se yeast as the appropriate level of Se enrichment during cabbage cultivation. These findings enhanced our understanding of the effects of Se yeast on the growth and quality of cabbage and provided new insights into Se-enrichment vegetable cultivation.

1. Introduction

Selenium (Se) is a trace element essential for human and animal health and has many functions. it acts as an anti-cancer agent, protects the heart, detoxifies the body, improves immunity, and delays aging. Se-deficient regions are widely distributed all over the world, including the Middle East, Finland, and some European countries.^1,2 China alone has as many as 22 Se-deficient provinces, and 72% of the counties and cities of the country are Se-deficient areas.^3,4 The daily dietary Se intake of nearly two-thirds of the people in China is 40 µg,⁵ which is lower than the WHO recommended daily Se intake of 55–400 µg for adults.⁶ Previous studies showed that Se deficiency can cause a series of health problems, such as Keshan disease, Kashin-Beck disease, and various cardiovascular diseases.³ Therefore, proper Se intake is of great significance for human health.

Se yeast is usually produced by cultivating specific yeast strains in a Se-enriched medium. As an organic Se source, it can convert inorganic Se into organic forms, such as selenocysteine (SeCys) and selenomethionine (SeMet). Using Se yeast as a bioaugmentation source of Se presents the advantages of low toxicity and high bioavailability. At present, Se yeast is widely used as an ideal Se-rich supplement in the feed industry to promote animal health and improve animal product quality. However, the application of Se yeast to the cultivation of Se-rich vegetables is rarely reported. Wu et al.⁷ found that the total protein and free amino acid content in Cardamine violifolia were significantly increased under 200 mg/L Se yeast treatment. The application of SeMet promoted the accumulation of beneficial substances and Se content in rice seedlings.⁸ Hu et al.⁹ found that the addition of organic Se can effectively block the accumulation of As in radish and promote the conversion of As from inorganic to organic form. Moreover, a study involving the combination of mycorrhiza technology and Se, it was found that mycorrhizal lettuces contained higher levels of proteins, sugars, and minerals than non-mycorrhizal ones when fertilized with Se.¹⁰

Brassica vegetables like cabbage are widely cultivated all over the world. It is rich in dietary fiber, minerals, carotenoids, vitamin C, folic acid, and glucosinolates. Documented results show that cabbage presents a certain Se enrichment ability. Appropriate levels of Se treatment can promote cabbage growth and quality, for instance, foliar application of 20 mg/L sodium selenite promotes Se accumulation in the leaves, heads, and roots of cabbage.¹¹ Rao et al.¹² also showed that Se can improve the fresh weight, free amino acids, and ascorbic acid content of Cardamine violifolia under Na₂SeO₄ treatment. Moreover, literature has been revealed that Se can modulate the level of phenolics and antioxidant enzyme activities in plants to alleviate the toxicity of abiotic and biotic stress.^13,14 A recent study showed that Se application is an efficient method to alleviate the deleterious effects of Chromium-stress and enhance the nutritional value by improving plant defense systems in Brassica juncea L. plants.¹⁵ However, current studies on Se-enriched cabbage are generally based on inorganic Se, which has safety risks. Se yeast is an ideal Se source candidate for Se-enrichment cultivation, it contains a large amount of organic Se (60%-80% as SeMet) and is safer and more efficient than inorganic Se.¹⁶ Cabbage can be used to transform organic Se from Se yeast, which may be of great significance for improving human health. The effect of Se yeast application on cabbage growth remains poorly understood. Therefore, three main aims were pursued in the present study. Firstly, the effects of Se yeast application on the accumulation of total Se and Se species of the cabbage head were determined. Secondly, the growth and nutritional quality changes of cabbage head under Se yeast treatments were determined. Thirdly, the effects of Se yeast on the beneficial secondary metabolites and antioxidant enzymes in the cabbage head were determined. The results of this study will provide new insights into Se-rich cabbage cultivation.

2. Materials and methods

2.1. Plant materials and treatments

Cabbage (Brassica oleracea var. capitata L. cv. ‘chunfeng’) is a hybrid of the two self-incompatibility cultivars, namely ‘Chicken Heart’ and ‘Jin Zaosheng’, which are bred by the vegetable Institute of Jiangsu Academy of Agricultural Sciences. It is mainly cultivated in South China. Cabbage seeds were sown to culture substrate (Se contents: 1.37 mg/kg DW) in hole trays. After 1 month, the seedlings were transplanted into plastic pots (diameter, 23.5 cm; height, 14 cm) containing 3.5 kg of the culture substrate per pot (pH 7.5). The doses of total nitrogen and phosphate fertilizers in culture substrate were 73.53 and 23.97 mg/kg, respectively. All pots with cabbage seedlings were cultivated in the greenhouse of Yangtze University, Jingzhou, China (30.35 °N, 112.14 °E). The Se yeast used in this study was purchased from Angel Yeast Co. (Yichang, China) with a total Se content of 2000 mg/kg.

Seven days after transplantation, 10 cabbage seedlings of uniform height were selected as the treatment group with three replicates. Se yeast was suspended in sterilized water and poured into the soil in the pots every 10 days. Se supplementation was performed four times, and the final Se concentrations in each pot were 0, 2, 4, 8, and 16 mg/kg (Se content per kilogram of culture substrate). Approximately 120 days after transplantation, cabbage head sizes, head weights, and total plant weights were measured. The plants were divided into roots, heads, and leaves, dried at 70°C, crushed, and then passed through a 40-mesh sieve for determination of Se contents. Fresh head leaves were frozen in liquid nitrogen and stored at – 80°C to determine other quality indices.

2.2. Measurement of total Se and Se species content in cabbage

Sample preparation for determining total Se content according to Rao et al.¹² with minor modification. Se contents were measured by liquid chromatography–atomic fluorescence spectrometry (LC-AFS8510, Beijing Haiguang, China). The Se standard sample (100 ug/mL) was purchased from the Chinese Academy of Metrology, and a standard curve was drawn. The instrumental conditions of LC-AFS are shown in Table S1.

The cabbage head (0.3 g) dried powders were mixed with 5 mL of protease XIV (4 mg/mL) and shaken overnight at 37°C, 200 rpm. After hydrolysis, the samples were centrifuged at 5000 g for 10 min at 4°C. The supernatant was filtered through 0.45-μm Millex (Nantong Supin Co.,) filters were subjected to Se speciation analysis by liquid chromatography–atomic fluorescence spectrometry (LC-AFS8510, Beijing Haiguang, China). Five single Se standard solutions [SeCys₂: selenocystine, SeMeCys: Se-methylselenocysteine, Se (IV): selenite, SeMet: selenomethionine, Se (VI): selenate] were configured as a 1000 µg/L mixed standard solution, and then diluted to 0, 20, 40, 60, 80, 100, 120,140, 160, 180 and 200 µg/L for drawing the standard curve. The identified Se species were quantitative and qualitative based on the peak areas and the retention time of standard compounds. The instrumental conditions of LC-AFS are shown in Table S1.

2.3. Determination of free amino acid content, soluble protein, soluble sugar, and chlorophyll content

Free amino acid, soluble protein, and soluble sugar were determined via the ninhydrin chromogenic method,¹³ coomassie brilliant blue G-250 method,¹⁷ anthrone colorimetric method,¹⁷ respectively. Chlorophyll contents were extracted with ethanol and determined by ultraviolet spectrophotometry.¹²

2.4. Determination of total glucosinolate, flavonoid, phenolic acid, and ascorbic acid content

Total glucosinolates content was determined according to the method of Tian et al.¹⁸ with an assay kit (Solarbio Biochemical Kit Division, China). The total flavonoid content of the cabbage head was determined by NaNO₂-Al (NO₃)₃ colorimetric method.¹⁹ The total phenolic acid was measured by FeCl₃-K₃(FeCN₆) coloration (Prussian Blue) with spectrophotometry according to Margraf et al.²⁰ Ascorbic acid was determined via the 2,6-dichloroindophenol titration method.¹²

2.5. Determination of antioxidant enzyme activity

Peroxidase (POD) activity was determined using an assay kit (Nanjing Jiancheng Biotechnology Co., Ltd, China), and superoxide dismutase (SOD) activity was determined using the nitrogen blue tetrazolium (NBT) method.²¹

2.6. Statistical analysis

The statistical analysis was performed using SPSS 24.0 (SPSS Inc., USA) software with Duncan’s new complex range test difference (p ≤ 0.05). All data are expressed as the mean ± SE of three replicates and plotted using SigmaPlot 14.0 (Systat Software Inc., USA).

3. Results

3.1. Effects of Se yeast on the growth of cabbage

The effects of different concentrations of Se yeast treatment on the growth performance of cabbage are shown in Figure 1 and Table 1. Compared with that of the control group, the size of cabbage heads treated with increasing concentrations of Se yeast gradually increased. The cabbage head size obtained under 16 mg/kg Se treatment was notably higher (25.4%) than that of the control, and the head weight of the 16 mg/kg was 88.3% higher than control. Total plant weights also significantly increased under Se yeast treatment. These results indicate that Se yeast treatment promoted the growth of cabbage.

Figure 1.

The head size of cabbage under different concentration of Se yeast treatments

Table 1.

The head size, head weights and total plant weights of cabbage under Se yeast treatment

Se levels (mg/kg)	Head size (cm)	Head weights (g)	Total plant weights (g)
0	11.4 ± 0.6b	303.33 ± 60.82b	539.47 ± 68.48b
2	12.23 ± 0.86ab	356.6 ± 68.25b	639.67 ± 22.88ab
4	13.1 ± 0.7ab	440.8 ± 81.97ab	723.63 ± 74.23a
8	12.3 ± 0.44ab	469.3 ± 44.91ab	772.74 ± 33.32a
16	14.3 ± 0.6a	571.13 ± 44.61a	800.48 ± 46.69a

The data are displayed with mean ± SE of three replications. Different letters indicate significant difference at p ≤ 0.05 among different Se level.

3.2. Effects of Se yeast on the total Se and Se species content of cabbage

As shown in Figure 2, Se contents in the roots, leaves, and heads of cabbage distinctly increased with the increasing Se yeast concentration. The highest value was observed in the 16 mg/kg Se yeast treatment. The maximum Se contents in cabbage roots, leaves, and heads reached 80.2, 84.42, and 100.55 mg/kg DW, respectively, which were 1231%, 2834%, and 3874% higher than the control. Besides, the Se contents of different plant organs showed the order roots > leaves > heads at Se yeast treatment concentrations below 16 mg/kg and the order heads > leaves > roots in the 16 mg/kg Se yeast treatment. These findings suggested that Se yeast treatment promotes Se accumulation in the roots, leaves, and heads of cabbage and that the distribution of Se in cabbage is tissue specific.

Figure 2.

The Se content in the roots, leaves, and heads of cabbage. The data are displayed with means ± SE of three replications. Different letters indicate a significant difference at p ≤ 0.05

In addition, MeSeCys was detected after treatment with Se yeast at 4, 8, 16 mg/kg and showed a significant difference under the different concentrations. The other three Se forms, namely, Se (IV), SeMet, and Se (VI), were not found. For 16 mg/kg Se yeast, the SeCys₂ and MeSeCys reached 1.38 and 2.69 μg/g, respectively (Table 2 and Figure 3). Moreover, SeCys₂ was detected in the 16 mg/kg Se treatment. These results showed that Se yeast treatment promotes the total Se and organic accumulation in the cabbage.

Table 2.

Se speciations in cabbage head treated with Se yeast

Treatment (mg/kg)	SeCys2 (μg/g)	MeSeCys (μg/g)
0	ND	ND
2	ND	ND
4	ND	0.360 ± 0.017 c
8	ND	0.992 ± 0.066b
16	1.380 ± 0.029	2.694 ± 0.189a

The data are displayed with the mean ± SE of three replications. Different letters indicate a significant difference at p ≤ 0.05.

ND: undetected; SeCys₂: selenocystine; SeMeCys: Se-methylselenocysteine.

Figure 3.

The isolation plot of standard Se substances by LC-AFS

SeCys₂: selenocystine, SeMeCys: Se-methylselenocysteine, Se (IV): selenite, SeMet: selenomethionine, Se (VI): selenate.

3.3. Effects of Se yeast on free amino acid, soluble protein, soluble sugar, and chlorophyll content of cabbage

Se yeast treatment significantly improved the nutrient quality in cabbage heads. as shown in Figure 4 A, the contents of free amino acids in cabbage heads significantly increased and peaked under 8 mg/kg Se yeast treatment. Soluble protein contents increased by treatment with 2 mg/kg Se yeast and then gradually declined with further increases in Se yeast concentration (Figure 4B). Se yeast treatments at above 4 mg/kg significantly enhanced soluble sugar contents in cabbage heads by 70.7%, 46.4%, and 74% (Figure 4C), respectively, compared with the control. Se yeast had a significant effect on the contents of chlorophyll in the cabbage head. Compared with the control, the contents of chlorophyll in cabbage heads treated with 2 mg/kg Se yeast increased by 54.3%. Further increases in Se treatment significantly decreased chlorophyll contents (Figure 4D). These results demonstrated that the application of Se yeast can promote the nutrient quality of cabbage.

Figure 4.

The content of free amino acid (A), soluble protein (B), soluble sugar (C), and chlorophyll (D) content in cabbage heads. The data are displayed with the mean ± SE of three replications. Different letters (a, b, c, d, e) indicate a significant difference at p ≤ 0.05

3.4. Effects of Se yeast on the content of total glucosinolate, flavonoid, phenolic acid, ascorbic acid, and the antioxidative enzyme activities of cabbage head

Effects of Se yeast on total glucosinolates, flavonoid, phenolic acids, ascorbic acid, and the antioxidative enzyme activities of cabbage head are shown in Figure 5. Compared with the control, 8 and 16 mg/kg Se yeast markedly improved the content of total glucosinolates by 44.8% and 65.1%, respectively (Figure 5A). However, no significant difference was observed below 4 mg/kg Se yeast compared with the control. Similarly, the highest amount of flavonoid content was found in the 0 mg/kg treatment (11.32 mg/g DW) followed by the 8 mg/kg treatment (11.2 mg/g DW). It was found that the content of total flavonoids was inhibited at low concentrations and enhanced at high concentrations (Figure 5B). Among the treatment concentrations, the highest total phenolic acid content was found in the 4 mg/kg treatment (35.15 mg/g DW) followed by 8 mg/kg (30.49 mg/g DW), which were 29.1% and 12.3% higher than the control, respectively (Figure 5C). Se yeast treatment promoted the accumulation of ascorbic acid in cabbage heads, although no significant difference between the treatment and control groups was observed (Figure 5D). Se yeast remarkably influenced the SOD activity of cabbage heads (Figure 5E). SOD activity in cabbage heads gradually increased with increasing Se treatment, peaked after application of 4 mg/kg Se yeast, which was 43.2% higher than the control, and then slightly decreased. POD activity decreased after treatment with 2 mg/kg Se yeast but increased and remained at relatively high levels by treatment with 4–8 mg/kg Se yeast treatment (Figure 5F). The above results indicate that Se yeast treatment could improve the antioxidative response, thus enhancing the stress resistance in the cabbage.

Figure 5.

The total glucosinolate content (A), total flavonoid content (B), total phenolic acid (C), ascorbic acid content (D), the superoxide dismutase (SOD) activity (E), and peroxidase (POD) activity (F) in cabbage heads. The data were expressed as the mean ±SE of three replicates. Different letters (a, b, c, d) indicate significant difference at p ≤ 0.05

4. Discussion

As an ideal organic Se carrier, Se yeast plays an important role in promoting animal health and improving the quality of animal products. However, little is known about the functions of Se yeast in the plant, especially in cabbage, which is a popular vegetable in China. In this study, we evaluated the effect of different concentrations of Se yeast treatment on cabbage growth performance and quality indices. We found that cabbage growth was significantly promoted by Se yeast, especially at high concentrations. The main Se forms in Se yeast are SeCys and SeMet, which are less toxic and more conducive to plant growth than inorganic.²² In the present study, Se contents in cabbage roots, heads, and leaves increased with increasing Se yeast concentration, which was consistent with the results of Mechora et al.²³ who used Se (IV) or Se (VI) on broccoli. Se could be transferred and redistributed to the leaves and reproductive organs of plants at high concentrations,²⁴ which fully explains the reason for Se content in cabbage head at 16 mg/kg is higher than that in leaves and roots. This finding implied that Se had a high transfer and accumulate efficiency in cabbage.

Various studies on the effect of Se species on vegetables, especially pakchoi,²⁵ have been conducted to understand the value of Se in human nutrition. As with the above-mentioned studies, the present study confirmed that some Se compounds in cabbage heads can also be released. Both SeCys₂ and MeSeCys were the main soluble species, but SeMet was not observed here. Although Se yeast contains a large amount of SeMet, the amount of SeMet that plants can absorb is limited. Excessive SeMet is easily retained in the roots and cannot be transported easily to the shoots.²⁶ MeSeCys, a kind of potent cancer-preventive substance, can be highly synthesized and accumulated by brassica plants.²⁷ Brassica plants can convert SeCys into MeSeCys and then into dimethyl diselenide (DMDSe) via SeCys methyltransferase (SMT) to achieve detoxification.⁷ In the present study, the main Se form in the cabbage head is MeSeCys, which means that cabbage can convert the Se from Se yeast into other Se forms.

Se yeast treatment effectively promoted cabbage quality, especially the free amino acid contents of cabbage heads, which consistent with the application of Se (IV) to B. juncea seedlings reported by Handa et al.¹³ It is probably due to the Se metabolized in plants is mainly in the form of amino acids, furthermore, SeMet and SeCys are the main Se species in the Se yeast. In the present study, soluble protein contents in cabbage heads increased at low Se concentrations but declined at high Se concentrations. Previously, a similar phenomenon was noticed in Brassica napus under different Se (IV) (0, 25, 50, and 100 mmol) treatments.¹⁷ Appropriate Se treatment can increase soluble protein contents, thereby suggesting the great ability of brassica plants to synthesize these osmolytes against Se phytotoxicity.²⁸ The improvement of soluble protein also may be due to Se yeast was absorbed by plants exists as amino acids, thus regulating the protein synthesis. Se yeast can induce soluble sugar accumulation to protect plants, as demonstrated by our results. The soluble sugars start accumulating under stressful conditions and play an important role in maintaining osmotic homeostasis, thereby further maintaining the integrity of various biomolecules and membranes. Se has similar effects on the content of soluble sugars in Brassica napus.¹⁷ The contents of chlorophyll of cabbage heads effectively increased at low concentrations and decreased with high concentrations of Se yeast. This change may be due to Se mainly metabolized in mesophyll cells in plant,²⁹ which inhibited the content of chlorophyll at high Se concentrations. Overall, the results implied that Se yeast treatment could improve cabbage quality.

Glucosinolates, flavonoids, and phenolic acid are secondary metabolites that have special research attention because of their importance in human health. Therefore, the combination of organic Se and these metabolites may have important research value. Recent studies on total glucosinolates of three different cabbage cultivars under Se treatment revealed that Se supply generally did not affect the total glucosinolate accumulation in Brassica sprouts.²⁷ In the present study, Se yeast treatment increased glucosinolate content, indicating that the effect of Se on the synthesis of glucosinolates in cabbage head might be related to the source of Se. The specific mechanism underlying Se yeast to cabbage still needs more research to reveal. Few pieces of literature have evaluated the effects of Se on flavonoid synthesis in cabbage. The present study discovered that Se yeast at low and high concentrations inhibited and promoted the synthesis of total flavonoids, respectively, but the values obtained were not significantly different from those obtained from the control. These results partially corresponded with those already available in the literature.³⁰ Perhaps Se exerts its antioxidant effect at low concentrations, thus, there is no need to accumulate a high amount of flavonoids to relieve the damage caused by active oxygen. In the present study, Se yeast treatment increased the content of total phenolic acid in the cabbage head, although the content was not significantly different from the control. The results of our experiment partially corresponded with those already available in the literature.³¹ The effects of Se on phenolic compounds varied depending on plant species, plant growth stage, and Se treatments.³² Therefore, the differences in this study may be due to these reasons.

Reactive oxygen species (ROS) are produced by plants in response to various environmental stresses. Ascorbic acid is an important antioxidant involved in ROS removal. Ren et al.³³ reported that Se treatment could increase the ascorbic acid content of Fuji apple, which is consistent with our study. SOD and POD are the main antioxidative enzymes involved in ROS elimination; thus, these enzymes protect plants from oxidative damage. Se can enhance antioxidant activities at low concentrations and promote oxidation at high concentrations.³⁴ Thus, the observed enhancements in plant growth by Se may be partly attributed to the enhanced antioxidant activity. In this study, Se treatment significantly enhanced SOD activity and maintained POD activity in cabbage, which was consistent with the results of Djanaguiraman et al.³⁵ These findings indicate that SOD plays a more important role than POD in ROS removal.³⁶ Taking the results together, Se yeast treatment enhances the antioxidant tolerance of cabbage.

5. Conclusion

In summary, Se yeast treatment effectively promoted cabbage growth, quality, and Se accumulation of cabbage, especially 8 mg/kg Se yeast treatment maintained high levels of important cabbage quality indices, such as the content of total Se, free amino acid, soluble sugar, ascorbic acid, phenolic acid, flavonoids, glucosinolates, and SOD activities. Therefore, the addition of 8 mg/kg Se yeast may be an efficient way to achieve Se-enriched cabbage. Human beings who consume 24 g to 170 g of fresh cabbage treated with 8 mg/kg Se yeast daily can meet the Se intake stipulated by WHO and obtain some important nutrients. Therefore, Se yeast is available as a source of Se biofortification in Se-enriched cabbage.

Funding Statement

This work was funded by the Hubei Technological Innovation Special fund (No. 2019ABA113) and the Key Research and Development Program of Hubei Province (No. 2020BBA043).

Author’s contribution

Feng Xu, Shuiyuan Cheng, and Shen Rao conceived the experiment. Xiaoli Liao wrote the draft of the manuscript and performed the article. Tian Yu, Zhenzhou Zhu, Hua Xue, and Xuefeng Wang contributed in determining the Se content. Xiaoyan Yang made a strict correction of the article. Yuanyuan Gou, contributed to collect the cabbage plants and determine the indexes. All authors have reviewed and approved the manuscript.

Disclosure statement

The authors declare that they have no competing interests.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website